Tonic inhibition determines the excitability of neurons and the activity of neuronal circuits through the persistent activity of specialized populations of high affinity extrasynaptic ?-aminobutyric acid A receptors (GABAARs), that are the principle targets of neurosteroids. Neurosteroids are widely accepted as endogenous regulators of GABAergic inhibition. Fluctuations in the levels of neurosteroids are accepted to play a critical role in epilepsy, and are also relevant to autism, anxiety, depression, premenstrual syndrome and schizophrenia. However, to date, there is a fundamental gap in understanding how fluctuations in the levels of neurosteroids change extrasynaptic GABAAR subunit expression levels, which are a significant factor in the changes in GABAergic inhibition that occur in a plethora of neuropsychiatric disorders. Our long-term goal is to fully understand the mechanism by which neurosteroids influence the expression levels of extrasynaptic GABAARs. In this proposal we will address the role that neurosteroids play in protein kinase C (PKC)-dependent phosphorylation of ?4 subunit-containing GABAARs. We will determine how this influences the cell surface accumulation of the GABAAR subtypes that mediate tonic inhibition. Our central hypothesis is that neurosteroids modulate the phosphorylation of GABAARs assembled from ?4?3 and ? subunits, which mediate the majority of tonic inhibition in the dentate gyrus and thalamus. Neurosteroids specifically potentiate PKC-dependent phosphorylation of serine 443 (S443) in the ?4 subunit. This increased phosphorylation leads to enhanced insertion of receptors composed of ?4?3 and ? subunits into the plasma membrane that originate within the secretory pathway. These enhancements of receptor cell surface stability are responsible for sustained increases in the efficacy of tonic inhibition. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: (1) Determining the role of PKC-dependent phosphorylation in modulating the specific cell surface accumulation of GABAARs that mediate tonic inhibition (2) Visualizing neurosteroid- mediated changes in cell surface stability and membrane trafficking of GABAARs and (3) Ascertaining the effects of neurosteroid-dependent phosphorylation on the activity of GABAARs and the efficacy of tonic inhibition. In summary this proposal will demonstrate a new and unexpected mechanism by which neurosteroids exert persistent and sustained changes in the efficacy of tonic inhibition. Understanding neurosteroid-mediated changes in phosphorylation and cell surface expression of GABAARs has the potential to translate into better understanding of neuropsychiatric disorders. Moreover such information is likely to lead to the development of more efficacious treatments for anxiety, depression, premenstrual syndrome, schizophrenia and substance abuse.